Packing materials for liquid chromatography (LC) are generally classified into two types: those having organic or polymeric carriers, e.g., polystyrene polymers; and those having inorganic carriers typified by silica gel. The polymeric materials are chemically stable against alkaline and acidic mobile phases; therefore, the pH range of the eluent used with polymeric chromatographic materials is wide, compared with the silica carriers. However, polymeric chromatographic materials generally result in columns having low efficiency, leading to inadequate separation performance, particularly with low molecular-weight analytes. Furthermore, polymeric chromatographic materials shrink and swell upon solvent changeover in the eluting solution.
On the other hand, silica gel-based chromatographic devices, e.g., HPLC columns, are most commonly used. The most common applications employ a silica which has been surface-derivatized with an organic functional group such as octadecyl (C18), octyl (C8), phenyl, amino, cyano group, etc. As a stationary phase for HPLC, these packing materials result in columns with high theoretical plate number/high efficiency, and do not evidence shrinking or swelling. Silica gel is characterized by the presence of silanol groups on its surface. During a typical derivatization process such as reaction with octadecyldimethylchlorosilane, at least 50% of the surface silanol groups remain unreacted.
A drawback with silica-based columns is their limited hydrolytic stability. First, the incomplete derivatization of the silica gel leaves a bare silica surface which can be readily dissolved under alkaline conditions, generally pH>8.0, leading to the subsequent collapse of the chromatographic bed. Secondly, the bonded phase can be stripped off of the surface under acidic conditions, generally pH<2.0, and eluted off the column by the mobile phase, causing loss of analyte retention, and an increase in the concentration of surface silanol groups. These problems have been attributed to free silanol group activity and hydrolytic instability of silica-based stationary phases. To address to these problem, many methods have been tried including use of ultrapure silica, carbonized silica, coating of the silica surface with polymeric materials, endcapping free silanol groups with a short-chain reagent such as trimethylchlorosilane, and the addition of suppressors such as amines to the eluent. These approaches have not proven to be completely satisfactory in practice.
Hybrid columns which combine organic and silica systems are known (XTerra™ MS C18 (Waters Corp., Milford, Mass. USA) and offer, potentially, the benefits of both silica and organic based materials. Hybrid particles have the advantages of both silica and polymer packing materials. In particular, hybrid particles offer mechanical strength, high efficiency, ability to separate a wide range of compounds, high chemical and temperature stability with little to no peak tailing, and improved peak shape for basic compounds. However, these materials have certain limitations, also.
Many of the limitations of hybrid silica-based columns can be attributed to surface organic (i.e., methyl groups). In particular, the presence of surface organic groups lead to lower bonded phase surface concentrations after bonding with silanes, e.g., C18 and C8 silanes, in comparison to silica phases, presumably because the methyl groups on the surface are unreactive to bonding. Further, in C18 bonded phases, surface organic groups may decrease the level of cross-bonding between adjacent C18 ligands. This results in reduced low pH stability since the C18 ligand has fewer covalent bonds to the surface of the particle. Ultimately, reduced retention times and peak compression can result from the reduced low pH stability caused by surface organic groups.